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1.
Oncotarget ; 6(6): 3932-46, 2015 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-25668816

RESUMO

HER2-positive breast tumors are associated with a high risk of brain relapse. HER3 is thought to be an indispensible signaling substrate for HER2 (encoded by ERBB2) and is induced in breast cancer-brain metastases, though the molecular mechanisms by which this oncogenic dimer promotes the development of brain metastases are still elusive. We studied the effects of the HER3-HER2 ligand, heregulin (neuregulin-1, broadly expressed in the brain), on luminal breast cancer cell lines in vitro. Treatment of SKBr3 (ERBB2-amplified), MDA-MB-361 (ERBB2-amplified, metastatic brain tumor-derived) and MCF7 (HER2-positive, not ERBB2-amplified) cells with exogenous heregulin increased proliferation and adhesive potential, concomitant with induction of cyclin D1 and ICAM-1, and suppression of p27. All three cell lines invaded through matrigel toward a heregulin chemotactic signal in transwell experiments, associated with activation of extracellular cathepsin B and matrix metalloproteinase-9 (MMP-9). Moreover, heregulin induced breast cancer cell transmigration across a tight barrier of primary human brain microvascular endothelia. This was dependent on the activity of HER2, HER3 and MMPs, and was completely abrogated by combination HER2-HER3 blockade using Herceptin® and the humanized HER3 monoclonal antibody, EV20. Collectively these data suggest mechanisms by which the HER3-HER2 dimer promotes development of metastatic tumors in the heregulin-rich brain microenvironment.


Assuntos
Barreira Hematoencefálica/metabolismo , Neoplasias da Mama/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neuregulina-1/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Barreira Hematoencefálica/enzimologia , Barreira Hematoencefálica/patologia , Encéfalo/irrigação sanguínea , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Feminino , Humanos , Células MCF-7 , Transdução de Sinais
2.
Cell Transplant ; 21(6): 1235-44, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22405378

RESUMO

The feasibility of ex vivo blood production is limited by both biological and engineering challenges. From an engineering perspective, these challenges include the significant volumes required to generate even a single unit of a blood product, as well as the correspondingly high protein consumption required for such large volume cultures. Membrane bioreactors, such as hollow fiber bioreactors (HFBRs), enable cell densities approximately 100-fold greater than traditional culture systems and therefore may enable a significant reduction in culture working volumes. As cultured cells, and larger molecules, are retained within a fraction of the system volume, via a semipermeable membrane it may be possible to reduce protein consumption by limiting supplementation to only this fraction. Typically, HFBRs are complex perfusion systems having total volumes incompatible with bench scale screening and optimization of stem cell-based cultures. In this article we describe the use of a simplified HFBR system to assess the feasibility of this technology to produce blood products from umbilical cord blood-derived CD34(+) hematopoietic stem progenitor cells (HSPCs). Unlike conventional HFBR systems used for protein manufacture, where cells are cultured in the extracapillary space, we have cultured cells in the intracapillary space, which is likely more compatible with the large-scale production of blood cell suspension cultures. Using this platform we direct HSPCs down the myeloid lineage, while targeting a 100-fold increase in cell density and the use of protein-free bulk medium. Our results demonstrate the potential of this system to deliver high cell densities, even in the absence of protein supplementation of the bulk medium.


Assuntos
Técnicas de Cultura de Células/métodos , Antígenos CD34/metabolismo , Reatores Biológicos , Antígeno CD11b/metabolismo , Técnicas de Cultura de Células/instrumentação , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Antígenos CD15/metabolismo
3.
Cytotherapy ; 13(3): 366-77, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20860426

RESUMO

BACKGROUND AIMS: Despite the availability of modern antibiotics/antimycotics and cytokine support, neutropenic infection accounts for the majority of chemotherapy-associated deaths. While transfusion support with donor neutrophils is possible, cost and complicated logistics make such an option unrealistic on a routine basis. A manufactured neutrophil product could enable routine prophylactic administration of neutrophils, preventing the onset of neutropenia and substantially reducing the risk of infection. We examined the use of pre-culture strategies and various cytokine/modulator combinations to improve neutrophil expansion from umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HPC). METHODS: Enriched UCB HPC were cultured using either two-phase pre-culture strategies or a single phase using various cytokine/modulator combinations. Outcome was assessed with respect to numerical expansion, cell morphology, granulation and respiratory burst activity. RESULTS: Pre-culture in the absence of strong differentiation signals (e.g. granulocyte colony-stimulating factor; G-CSF) failed to provide any improvement to final neutrophil yields. Similarly, removal of differentiating cells during pre-culture failed to improve neutrophil yields to an appreciable extent. Of the cytokine/modulator combinations, the addition of granulocyte-macrophage (GM)-colony-stimulating factor (CSF) alone gave the greatest increase. In order to avoid production of monocytes, it was necessary to remove GM-CSF on day 5. Using this strategy, neutrophil expansion improved 2.7-fold. CONCLUSIONS: Although all cytokines and culture strategies employed have been reported previously to enhance HPC expansion, we found that the addition of GM-CSF alone was sufficient to improve total cell yields maximally. The need to remove GM-CSF on day 5 to avoid monocyte differentiation highlights the context and time-dependent complexity of exogenous signaling in hematopoietic cell differentiation and growth.


Assuntos
Antígenos CD34/metabolismo , Sangue Fetal/citologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/farmacologia , Esterases/metabolismo , Etilenodiaminas/farmacologia , Heparina/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Neutrófilos/enzimologia , Oxigênio/farmacologia , Superóxidos/metabolismo , Tacrolimo/farmacologia , Fatores de Tempo , Ácido Valproico/farmacologia
4.
Biotechnol Bioeng ; 104(4): 832-40, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19591208

RESUMO

Dose-intensive chemotherapy results in an obligatory period of severe neutropenia during which patients are at high risk of infection. While patient support with donor neutrophils is possible, this option is restricted due to donor availability and logistic complications. To overcome these problems, we explored the possibility of large scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells (HPC). CD34+ HPC isolated from umbilical cord blood (UCB) and mobilized peripheral blood (mPB) were expanded in serum-free medium supplemented with stem cell factor, granulocyte colony stimulating factor, and a thrombopoietin peptide mimetic. After 15 days of cultivation a 5,800-fold expansion in cell number was achieved for UCB, and up to 4,000-fold for mPB, comprising 40% and 60% mature neutrophils respectively. Ex vivo expanded neutrophils exhibited respiratory burst activity similar to that for donor neutrophils, and were capable of killing Candida albicans in vitro. These yields correspond to a more than 10-fold improvement over current methods, and are sufficient for the production of multiple neutrophil transfusion doses per HPC donation. To enable clinical scale manufacture, we adapted our protocol for use in a wave-type bioreactor at a volume of 10 L. This is the first demonstration of a large scale bioprocess suitable for routine manufacture of a mature blood cell product from HPC, and could enable prophylactic neutrophil support for chemotherapy patients.


Assuntos
Técnicas de Cultura de Células/métodos , Sangue Fetal/citologia , Células-Tronco Hematopoéticas , Neutrófilos/fisiologia , Adulto , Candida albicans/imunologia , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Meios de Cultura Livres de Soro , Humanos , Viabilidade Microbiana , Neutrófilos/imunologia
5.
Bioorg Med Chem ; 13(6): 2065-77, 2005 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15727860

RESUMO

2-Alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones (F(2)-NH-DABOs) 4, 5 belonging to the dihydro-alkoxy-benzyl-oxopyrimidine (DABO) family and bearing different alkyl- and arylamino side chains at the C(2)-position of the pyrimidine ring were designed as active against wild type (wt) human immunodeficiency virus type 1 (HIV-1) and some relevant HIV-1 mutants. Biological evaluation indicated the importance of the further anchor point of compounds 4, 5 into the non-nucleoside binding site (NNBS): newly synthesized compounds were highly active against both wild type and the Y181C HIV-1 strains. In anti-wt HIV-1 assay the potency of amino derivatives did not depend on the size or shape of the C(2)-amino side chain, but it associated with the presence of one or two methyl groups (one at the pyrimidine C(5)-position and the other at the benzylic carbon), being thymine, alpha-methyluracil or alpha-methylthymine derivatives almost equally active in reducing wt HIV-1-induced cytopathogenicity in MT-4 cells. Against the Y181C mutant strain, 2,6-difluorobenzyl-alpha-methylthymine derivatives 4d, 5h'-n' showed the highest potency and selectivity among tested compounds, both a properly sized C(2)-NH side chain and the presence of two methyl groups (at C(5) and benzylic positions) being crucial for high antiviral action.


Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Compostos de Benzil/química , Compostos de Benzil/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/classificação , Alquilação , Aminação , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/classificação , Linhagem Celular , Flúor/química , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Mutação/genética , Nucleosídeos/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-Atividade
6.
J Med Chem ; 47(4): 928-34, 2004 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-14761194

RESUMO

Dihydro-alkoxy-benzyl-oxopyrimidines (DABOs) are a family of potent NNRTIs developed in the past decade. Attempts to improve their potency and selectivity led to thio-DABOs (S-DABOs), DATNOs, and difluoro-thio-DABOs (F(2)-S-DABOs). More recently, we reported the synthesis and molecular modeling studies of a novel conformationally constrained subtype of the S-DABO series characterized by the presence of substituents on the methylene linkage connecting the pyrimidine ring to the aryl moiety (Mai, A., et al. J. Med. Chem. 2001, 44, 2544-2554). Now we report the computer-aided design, synthesis, and biological evaluation of four new DABO prototypes (5-alkyl-2-cyclopentylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydropyrimidin-4(3H)-ones, F(2)-NH-DABOs) in which the sulfur atom of the related F(2)-S-DABOs is replaced by an amino group. For these studies, we used as a reference model the cocrystallized MKC-442/RT complex. Docking studies with Autodock of the newly designed F(2)-NH-DABOs on the ligand-derived RT confirmed the findings previously described for the F(2)-S-DABOs. The F(2)-NH-DABO binding mode resembles that reported for F(2)-S-DABOs, with the difference that the NH moiety at the C-2 position represents a new anchor site for ligand/enzyme complexation. The predicted inhibition constant (K(i)) values by the internal scoring function of Autodock, and the predicted IC(50) values by the application of a VALIDATE II/HIV-RT model strongly suggested the synthesis of the designed amino-DABOs. F(2)-NH-DABOs were shown to be highly active in both anti-RT and anti-HIV biological assays with IC(50) and EC(50) comparable with that of the reference compound MKC-442. Interestingly, 2-cyclopentylamino-6-[1-(2,6-difluorophenyl)ethyl]-3,4-dihydro-5-methyl pyrimidin-4(3H)-one (9d) was active against the Y181C HIV-1 mutant strain at submicromolar concentration, with a resistance value similar to that of efavirenz, the last FDA-approved NNRTI for AIDS therapy, and 2-fold lower than that of its 2-cyclopentylthio counterpart 8d. The introduction in 9d of a new anchor point (pyrimidine C-2 NH group), with the formation of a new hydrogen bond with Lys101, could compensate for the lack of positive hydrophobic ligand/NNBP interactions due to the Tyr181 to Cys181 mutation.


Assuntos
Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , Pirimidinonas/síntese química , Inibidores da Transcriptase Reversa/síntese química , Linhagem Celular , Simulação por Computador , Desenho de Fármacos , Farmacorresistência Viral , HIV-1/genética , HIV-2/efeitos dos fármacos , Humanos , Modelos Moleculares , Mutação , Pirimidinonas/química , Pirimidinonas/farmacologia , Relação Quantitativa Estrutura-Atividade , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia
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